Eur. J. Biochem. 87, 9- 19 (1978)

Interchangeable Copper and Iron Proteins in Algal Photosynthesis Studies on Plastocyanin and Cytochrome c-552 in Chlamydomonas Paul M. WOOD Department of Biochemistry, University of Cambridge (Received December 1, 1977)

The interrelation of the copper protein plastocyanin, and a soluble c-type cytochrome, c-552, in photosynthetic electron transport has been studied in the genus Chlamydomonas. With C. reinhardtii the plastocyanin : cytochrome c-552 ratio could be changed from 300: 1 to < 1 : 16 simply by omitting copper from the medium, without any other detectable change. Plastocyanin was indetectable in a second species, C. mundana, for which the cytochrome c-552 level was always very high. The properties of Levine’s C. reinhardtii mutant lacking plastocyanin, ac-208, were studied and it was found that the photosynthetic capabilities of a suppressed phenotype and suppressed genotype could be explained by reference to the cytochrome c-552 levels. Both proteins were successfully used in reconstitution experiments with chloroplast fragments. Both showed very fast kinetics for reduction by purified Chlamydomonas cytochromef, but the rate of electron transfer from one to the other was much slower. It is concluded that they constitute an interchangeable pair, and the rationale for this and possible analogies are both discussed. There are three well-characterised proteins with roles in the electron transport chain of oxygenic photosynthesis between plastoquinone and P700. The first is cytochrome f , typified by the protein purified from parsley by Hill and Scarisbrick [l]. Cytochrome f is an intrinsic protein, and can only be extracted and purified by total disruption of the membranes. The molecular weight in dodecylsulphate is about 33000 [2,3] and the y-band maximum is at the high value of 421 -422 nm. The second is the photosynthetic soluble c-type cytochrome (c-552, c-553 or c-554; here generally referred to as c-552) which seems universal in the algae studied but is unknown in higher plants (see [4] for a list of sources; it may be significant that this does not include any of the more advanced green algae). The molecular weight is about 11000 and the y-band at about 416 nm. The third protein is plastocyanin, a copper protein with molecular weight and redox potential resembling cytochrome c-552, and likewise largely released whenever the thylakoids are broken. It is universal in higher plants, widespread in green algae, present in some blue-greens, but has so far not been isolated from any other algal type [4]. In the past it has frequently been assumed that cytochrome c-552 is the algal equivalent of higher plant cytochrome f , and that membrane-bound and soluble fractions were simply forms of the same -~

Abbreviation. P700, reaction centre of photosystem 1

protein and had the same function [4]. But recently a cytochrome has been extracted from Chlamydomonas, Euglena and Anacystis which resembles parsley cytochrome f and differs from cytochrome c-552 in molecular weight, position of y-band, shape of b-band, rate of electron transfer to plastocyanin, and behaviour with cytochrome c-552 antiserum [S]. It seems logical to denote it (and it alone) by cytochromef, and this is the convention adopted here. The same procedure has since yielded a similar product from Scenedesmus and Bumilleropsis (a diatom) [6], so cytochrome f is certainly widely distributed, and may well be universal in algae. In higher plants the now widely accepted order of the carriers is plastoquinone - cytochrome f - plastocyanin- P700 [7]. After removal of plastocyanin, reconstitution is also possible with Euglena cytochrome c-552 [8]. Wildner and Hauska showed that for Euglena, conversely, one could reconstitute with higher plant plastocyanin [9]. It is likely that in Euglena the carriers are plastoquinone - cytochrome f - cytochrome c-552 - P700 [4,5,10]. This closely resembles part of the mitochondria1 chain, since the haem peptide of cytochrome c1 has a molecular weight of 31 000 in dodecylsulphate and an cr-band at 553.5 nm when purified from yeast [Ill, while mitochondrial cytochrome c-550 shows sequence homology with algal cytochrome c-552 [12]. By comparison it would appear that in the course of evotution from the first blue-green algae to higher plants the soluble

10

cytochrome c has been replaced by plastocyanin [41. But there are also many species that contain both the soluble cytochrome c and plastocyanin, and at present these comprise a wide range of green algae as well as some blue-greens [4]. The implication is that in these algae both proteins have the same role, and indeed both have successfully been used in reconstitutions, first with Anabaenu in 1967 [13] and later with Scenedesmus [14]. However the interpretation of these results has frequently been blurred by a belief that the cytochrome in addition has the function of higher plant cytochrome f. If one removes this problem, the two proteins are left as exactly equivalent, an unusual situation. This paper examines their interrelation in Chlamydomonas, and makes use of the conventional C. reinhardtii, a contrasted species, C. mundana, and a mutant lacking plastocyanin, C. reinhardfii ac-208. C. mundana Gerloff, 'Boron' strain was chosen for comparison because this strain was originally isolated from an anaerobic sewage pond with free H2S, in which it was able to form blooms under conditions where one would expect copper to be trapped in insoluble sulphides [15- 171. C. reinhardtii ac-208 was isolated by Levine and coworkers, and shown by them to lack plastocyanin but have no other detectable deficiency [18]. The best proof was reconstitution of electron transport in its chloroplast fragments by added plastocyanin. Gorman and Levine found that mutant ac-208 gave rise to suppressor mutations [to ac-208 (sup.)] more readily than any other of their mutants, leading to a recovery of appreciable photosynthetic activity [18,19]. They were unable to trace the mechanism of this suppression, and postulated a synthesis of a particularly unstable plastocyanin, which was destroyed by the acetone extraction used to recover soluble proteins. They regarded the soluble cytochrome as a form of the bound cytochrome (cytochrome ,fi which happened to accumulate rather erratically towards the end of log phase; they did not investigate its level in mutant ac-208. Hence mutants ac-208 and ac-208(sup.) seemed an important testingground for theories about the role of cytochrome c-552, as well as being likely to give insight into regulatory mechanisms if the results supported the hypothesis of identical roles. Levine's mutant lacking cytochrome c-552, ac-206 [18,19] was not tried, since it is now clear that it also lacks cytochrome f, and therefore suppression or reconstitution raises altogether different problems [4,51. MATERIALS AND METHODS C . reinhardtii 137c (wild type) was obtained from Dr W. T. Griffiths (Dept. of Biochemistry, University

Plastocyanin and Cytochrome c-552 Interrelation

of Bristol). C. reinhardtii, mutant ac-208, was a kind gift from Prof. R. P. Levine (Harvard). C. mundana, 'Boron' strain, was obtained from the algal collection at Gottingen. A common growth medium was devised from earlier ones for these species [20,21] : 20 mM Tris, 3 mM K2HP04, 7 mM NH4C1, acetic acid to give pH 7, 100 pM H3BO3, 200 pM MgS04 . 7 HzO, 200 pM CaC12 . 2 € 1 2 0 , 220 pM NaZEDTA, 100 pM FeS04. 7 H20,60 jIM Z n S 0 4 .7 H20,25 pM MnCL . 4 H 2 0 , 8 pM C U S O ~ . 5 H 2 0 , 7 pM COC12 . 6 Hz0, 1 pM ammonium molybdate. The pH was finally readjusted to 7.0. For C. mundana vitamins BI and B12were added at 5 and 2.5 pg/l [21]. For autotrophic growth the Tris and acetic acid were omitted, and pH adjustments made with HCl and NaOH. For studies with copper omitted, all glassware was rinsed with 10 M HC1 and then with glass-distilled water, glassdistilled water was used throughout, and the inoculum, if from a normal medium, was spun down in a sterile test tube. Stocks were maintained on agar slopes, or small liquid cultures. Cultures were grown with continuous illumination (2000 - 3000 lux) at 25 "C, in 500 ml-2 1 flasks on an orbital shaker (80- 100 rev./ min), or with bottom stirring provided by a magnet encased in plastic. For mutant ac-208 a lower light level, 500- 1000 lux, was optimal, since growth in the dark was very slow, while high light intensities resulted in photodestruction. Suppressor mutations were troublesome, so photosynthetic oxygen evolution was tested frequently (see below), and any culture giving net evolution rather than reduced uptake was discarded. The strain of mutant ac-208(sup.) used was recovered from a culture which had been grown autotrophically for several weeks. In the course of several months experimentation its properties were completely stable, and it was normally grown at the same light intensity as the wild type. For chlorophyll assays the material was added to acetone and hand homogenised. Water was then added to give a final acetone concentration of SO%, the solution was centrifuged, and the absorption of the supernatant measured at 652 nm [22]. Measurements of oxygen evolution or uptake were made with an oxygen electrode (Hansatech, King's Lynn), with light provided by a slide projector fitted with a red filter (Schott RG610). Difference spectra were recorded with a sensitive split-beam spectrophotometer. Kinetic measurements involving oxidation of a cytochrome were monitored with a dual wavelength recording spectrophotometer, the cuvette being fitted with an efficient stirrer [23]. For complete ex1raction of soluble proteins (plastocyanin and cytochromes c-552 and c-550) the cells were spun down, washed in 10 mM phosphate pH 7, and frozen at - 80 "C in 10 mM phosphate, pH 7 plus 0.4 M NaC1. Thawing and centrifugation resulted in almost complete release of these proteins into the

P. M. Wood

Fig. 1. Oxidised minus reduced d f f i r m c e spectru f r o m 600 to 720 nm. The upper trace is for plastocyanin and the lower one for cytochrome c-552, both purified from Chlamydomonas reinhurdtii

supernatant; the last traces were recovered by washing the pellet with NaCl/phosphate, refreezing it in this medium, thawing, centrifuging, and pooling all supernatants. The chlorophyll in the pellet was assayed as above. The pooled material was dialysed against 10mM phosphate pH 7, and applied to a small column of DEAE-cellulose equilibrated with 10 mM phosphate, after adding a little ascorbate [24]. The basic cytochrome c-550 was not absorbed, and was assayed from the direct eluate by an ascorbate-ferricyanide difference spectrum, assuming A E = 19 mM cm-' at 550 nm relative to a line through 541.5 and 556.5 nm [25]. After washing the column with 10 mM phosphate (pH 7), plastocyanin and cytochrome c-552 were eluted together by 50 mM phosphate plus 0.15 M KC1 [24], and assayed by difference spectra. For cytochrome c-552, A s = 20 mM-' cm-' was assumed for the peak at 552.5 nm relative to a line through the isosbestic points at 542 and 561 nm [26]. Plastocyanin was assayed by nm nm = 2.8 mMP' cm-t derived from a difference spectrum for the purified protein, assuming A s = 4.5 mM-' cm-' at 597 nm [27]. When cytochrome c-552 was in excess over plastocyanin, it was necessary to compensate for absorption by the cytochrome in this region. Fig. 1 shows difference spectra for purified plastocyanin and cytochrome c-552 in the 600- 700 nm range; the spectrum of the oxidised cytochrome showed a slight peak at 690 nm and a shoulder at 640 nm, as reported for Euglena cytochrome c-552 [28]. The correction applied was A6615 n m - As680 n m = 0.47 mM-' cm-' for the cytochrome, and 1 mol plastocyanin per 25 - 30 mol cytochrome c-552 was the practical limit of detectability. Cytochrome c-550 oxidase activity was assayed with horse heart cytochrome c, freshly reduced with ascorbate and separated from excess reductant by Sephadex G-25. The algal cells for the assay were frozen, thawed and centrifuged, to remove endogenous

11

cytochrome c-550, and then resuspended in buffer, sonicated, and assayed for chlorophyll. Chloroplast fragments for reconstitution experiments were prepared by ultrasonic disruption of the cells at 0 "C in 10 mM phosphate, pH 7, plus 20 mM KCl and 2.5 mM MgClZ [29]. The sonicate was centrifuged for 3 min at 1000 x g, and the supernatant for 20 min at 20 000 x g. The pellet was resuspended in the same medium. Cytochrome f was purified from C. reinhardtii as described previously [5]. For purification of plastocyanin or cytochrome c-552 it was advisable to start with growth conditions under which the synthesis of the other was almost entirely repressed, as described below, since otherwise the later stages consisted largely of a separation of one from the other. Suitable conditions were, for plastocyanin, a full medium with efficient aeration, and for cytochrome c-552, a medium with no added copper. The cells were harvested, washed in 10 mM phosphate pH 7, and broken by freezing in this medium at - 80 "C. The supernatant remaining after centrifugation of the frozen and thawed cells was applied directly to a column of DEAE-cellulose, after addition of a pinch of ascorbate, and the purification continued as described by Gorman and Levine ~41. RESULTS Properties of C. reinhardtii (wild type) and C . mundana

Plastocyanin, cytochrome c-552 and the mitochondrial cytochrome c-550 were released together for assay by freezing in 0.4 M NaCl (buffered at pH 7), since low salt media [5] only gave partial extraction of cytochrome c-550 (cf. broken animal mitochondria) making it hard to be sure that no cytochrome c-552 remained. No further extraction occurred if the final pellet was sonicated or treated with acetone followed by aqueous buffer. Very little other coloured material was solubilised, while the plastocyanin levels reported below are substantially higher than those of an earlier study using acetone [IS]. All three proteins were insensitive to freezing. Cytochrome c-550 was separated from the others by chromatography after dialysis, but since the elution peaks of the other two overlapped in a salt gradient [26], they were assayed together. For C. reinhardtii, wild type, the contents of plastocyanin and cytochrome c-552 on a chlorophyll basis varied wildly, as Fig. 2 makes clear. (The level of cytochrome f showed little variation [S].) A variety of media and cultural conditions was tried and the levels of these proteins were found to be little affected by different starting pH values, iron concentrations, or by autotrophic growth (which lowered cytochrome c-550). Only two parameters were found to be im-

Plastocjanin and Cytochrome c-552 Interrelation

12

portant, the level of copper and the state of aeration. Three growth conditions were selected as giving the most disparate responses : growth on an orbital shaker plus added copper; growth on an orbital shaker but with copper omitted; growth with slow basal stirring. The results are shown in Table 1. With the first growth regime the plastocyanin level was very high, about 1 mol per 150 mol chlorophyll. Cytochrome c-552 was scarcely measurable, although a trace was always found, and did not increase at the end of log phase or even if the culture was left for a period of weeks. But if copper was omitted, plastocyanin became virtually indetectable, while a hundred-fold increase in cytochrome c-552 occurred. The final yield in mg chlorophyll was not affected, nor was growth significantly

Fig. 2. Contrasting difference spectra Jbr the plastocyanin plus cytochrome c-552 fraction from the DEAE-cellulose column. (I) C. reinhardtii grown in aerated full medium. (11) As I, but with copper omitted from the medium

slowed. Incubation of the supernatant from freezing and thawing with 50 pM CuSO4 for 30 min did not lead to a higher plastocyanin content, as would be expected if apoplastocyanin had accumulated in the cells [30]; there was also no spurt of plastocyanin synthesis on addition of copper to a copper-deficient culture. 0.5 pM Cu2+ added to the growth medium changed the response to that seen with copper present from the start, even with [EDTA] increased to 400 pM. For the third regime the full medium was used, but the cells were grown in fairly large, well-filled flasks, with slow bottom stirring. This led to a large increase in cytochrome c-552 in the later stages of growth, up to about 1 mol per 600 mol chlorophyll, with a slight decrease in plastocyanin, though the cytochrome never overtook it. A similar effect occurred when flasks on the shaker were sealed with a rubber bung, but the cultures often did not grow well. The build up of cytochr-ome c-552 was found to coincide with the culture becoming depleted of oxygen, which fell to < 10 % of the air-saturated level. This was partly attributable to a decline in photosynthetic oxygen evolution towards the end of log phase [14,31], but was more a question of self-shielding of most cells from the light. The turbulent motion on the orbital shaker gave rapid equilibration of dissolved oxygen with the air above, but slow basal stirring resulted in laminar flow and slow downwards movement of atmospheric oxygen through the culture. The stimulated cytochrome synthesis still occurred with 80 pM added copper (Table l), an equivalent amount of EDTA being added so that chelated and unchelated concentrations of other metals would be unchanged. The ambient fedox potential measured with a platinum electrode fell to Eh = + 150 mV in the later stages; but Cu . EDTA has Ern,, = + 130 mV [32], and indeed

Table 1. C. reinhardtii (wild type) and C. mundana under dijferent growth conditions The cells were harvested at the onset of stationary phase in (iii), and in the later stages of log phass (or later) for (i) and (ii). For cytochrome c oxidase the units are, pmol cytochrome c oxidised x mg chlorophyll-' x h-', with 2 pM reduced horse heart cytochrome c, and cell fragments disrupted by freezing and sonication. The buffer was 10 mM phosphate, pH 7, plus 80 mM NaC1, arid 540 and 550 nm were the two wavelengths on the spectrophotometer. n.d., not detected Conditions

C. reinhardtii, wild type (i) Aerated, plus Cu (ii) Aerated, n o added Cu (iii) Slow basal stirring, plus c u As (iii), but with 80 pM CU . EDTA C. mundana All conditions

Cytochrome c-552

Cytochrome c-550

Chlorophyll

Plastocyanin

mg/l

mo1/100000 mol chlorophyll

21 rfr 3 20 f 3

650 k I00 5 10

1.4 f 0.8 165 rfr 20

33 35

k3

350 f 100

140 f 40

40 rfr 8

13

480

230

54

12 f 2

n.d.

600 rfr 100

17

Cytochrome c oxidase pmol x mg-' x h-'

rfr 5 8

4f2

2.0 2.0

0.17

P. M. Wood

the medium was still blue after the algae had been harvested. There are analogies to these results in earlier work. The level of cytochrome c-552 in growth with added copper is very similar to that found by Gorman and Levine during log phase [26]. But under their conditions it built up in the later stages, reaching a variable level of 1 mol per 1000 to 10000 mol chlorophyll in stationary phase [26]. They used 12-1 volumes and agitation by aeration [24], and the cytochrome c-552 increase probably parallels that seen in growth with basal stirring; certainly the oxygen demand from a dense culture of this size would be very great. The experience of Kunert et a]. with unstirred Scenedesmus is also very similar [14]. As for the response to copper deficiency, this closely parallels Boger’s results in an independent series of experiments with Scenedesmus [6], though the present results do not suggest a fixed stoichiometry. C. mundana gave a very high yield of cytochrome c-552 under all cultural conditions, without much variation. (In this it resembled Euglena [9,10].) Plastocyanin was never detected, and a limit of < 1 mol per 30 mol cytochrome c-552 could be set. Cytochrome c-550 was present in very low amounts, perhaps reflecting the adaptation to anaerobic growth, but was definitely detected. C. mundana seems to be the first documented example of a green alga in which a thorough search has failed to find plastocyanin. The published media seldom contain copper [21]. For investigating the response of other copper proteins to omission of copper from the medium, the cyanide-sensitive copper-zinc superoxide dismutase might seem a natural choice. However in algae it has mysteriously only been found in the more advanced Chlorophyta [33], and a mitochondria1 protein was therefore used instead, cytochrome aa3, present as cytochrome c-550 oxidase. Table 1 shows its activity in membrane fragments, assayed by reduced horse heart cytochrome c. The omission of copper made no difference, while the relative activity correlated well with the level cytochrome of c-550. It should be noted that a copper concentration of about 100 nM is likely to be present from impurities, sinck reagent grade chemicals contain copper at 1- 10 parts per million and glass distilled water contains copper at 0.001 0.004 parts per million [34]. The specific response to added copper was ideal for turnover experiments, transferring algae from a medium minus copper to one plus copper, or vice versa. These showed a slight lag before response to the change followed by a slow turnover with a rather variable time constant of several days. The yields on harvesting (Table 1 and Table 3, below) consequently reflected synthesis throughout the history of a culture, a particularly important consideration in the basal stirring experiments, and many generations of growth

13 Table 2. Rates o j oxygen evolution by C . reinhardtii strains grown in aerated medium plus copper, and ej’ects of inhibitors The cells were harvested in the later stages of log phase, washed and resuspended in 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulphonate plus 10 mM KHC03, adjusted to pH 7.6. The same medium was used for assays, on the oxygen electrode with 25 pg chlorophyll/ml. Positive rates are for 0 2 evolution, negative values for uptake System

Rate with strain .__ __ wild type ac-208

~~

ac-Z08(sup )

pmol x mg chlorophyll-’ x h-’ ~-

- 22 (a) Dark + 150 (b) Illuminated (c) DiHerence, (b) - (a) +172(,40) (d) + 1 mM KCN, illuminated minus dark + 15 (e) + 1 I.IM 2,s-dibromomethylisopropyl-pbenzoquinone, illuminated + 6 minusdark

- 21 -

+

15 6(+_2)

- 21

+ 75 +96(+_30)

+ 3

+ 30

+ 2

+ 4

were needed to dilute out a previously dominant component. The slow turnover is in agreement with most other studies on chloroplast proteins [35]; chloroplasts from seaweed were found to be active three months after ingestion by sea-slugs, so turnover of proteins encoded by nuclear DNA is not necessary for function [19,36].

Properties of C . reinhardtii ac-208 and ac-208(sup.) Table 2 shows the capabilities of mutant ac-208 and wild type for photosynthetic oxygen evolution, both having been grown in an aerated medium plus copper. Various assay media were tried, including Warburg’s No. 9, but none gave higher rates. Whole cells were used, despite the complications of photorespiration, since Chlamydomonas contains a single large chloroplast which cannot be isolated intact, and all methods for preparing chloroplast fragments liberate appreciable amounts of plastocyanin and cytochrome c-552. For mutant ac-208, while a net oxygen uptake always occurred during illumination, the rate was definitely lower than in the dark. The change in rate on illumination was a very small fraction of that for wild type, but was inhibited in the normal way by KCN (which blocks ribulose bisphosphate carboxylase [37]) and 2,5-dibromomethyIisopropyl-p-benzoquinone (which inhibits between plastoquinone and cytochromefl. The faster growth of mutant ac-208 in dim light than in darkness and the inhibition by 2,5-dibromornethylisopropyl-p-benzoquinone imply that electron transport to P700 is taking place, but at a very slow rate.

Plastocyanin and Cytochrome c-552 Interrelation

14

Table 3. Photosynthetic oxygen evolution and protein levels in C. reinhardtii ac-208 and ac-208(sup.) Oxygen evolution was measured with whole cells as in Table 2. The cytochromes were assayed as in Table 1. Plastocyanin was not detected System

(a) ac-208, standard growth conditions (i) aerated, plus c u (ii) aerated, no added Cu (iiij slow basal stirring. plus CU (b) Diluting out the suppressed phenotype [(ii) and (iii)] Medium as (i), 5 m1/300 ml inoculum from (ii) Medium as (I), 2 m1/300 ml inoculum from (iii)

(c) Suppressed genotype, ac-208(sup.j (i) aerated. plus Cu (ii) aerated, no added Cu (iii) slow basal stirring, plus CU

0 2

evolution

Cytochrome c-552

Cytochrome c-550

pmol Oz x mg chlorophyll-' x h-'

mo1/100000 mot chlorophyll

5 1 2 90 & 20 110 & 40

2.4 0.6 150 20 1'70 & 50

+

*

95 & 30

*

34 12 26 k 10 2 6 i 6

21 22

9 9

24 30

Despite a careful search, plastocyanin was never detected under any conditions, and a limit of < 1 mol per 100000 mol chlorophyll could be set (cf. the earlier limit of < 1 mol per 2 x lo4 mol chlorophyll [ls]). Incubation of the supernatant from freezing and thawing with 50 pM Cu2+ showed no sign of apoprotein. Table 3 presents data for the three growth regimes used for wild type. The first regime, as used in Table 2, gave a very low cytochrome c-552 level, as in wild type. But either of the regimes which stimulated cytochrome c-552 synthesis in wild type caused a large recovery of oxygen evolution, together with a large increase in cytochrome c-552. Table 3 also shows that this development of a suppressed phenotype (not previously detected) was gradually reversed by subsequent growth in aerated medium plus copper. Several generations were required to dilute out the cytochrome c-552;cf. the remarks above on turnover in wild type. The levels of cytochrome c-550(Table 3) and cytochrome oxidase were normal, so the lesion does not affect all copper proteins. Cytochrome f was also normal (as in [18]). As for mutant ac-208(sup.),Table 2 shows that the rate of photosynthetic oxygen evolution was about half that of wild type, making it similar to the suppressed strains studied by Gorman and Levine [18]. Its cytochrome c-552 content (Table 3) proved to be considerably higher than that of unsuppressed cytochrome ac-208 grown in aerated medium plus copper. Unlike the unsuppressed form, the cytochrome c-552 level was not altered by omission of copper from the medium. With slow basal stirring it grew noticeably less well than the other strains; in healthy cultures the cytochrome c-552 level was just as in aerated growth. Other suppressor mutations appeared from time to time in the course of work with mutant ac-208, being detected by net oxygen evolution on illumina-

__

~~

L9+ 21 21

*

k 10

2

60

4

50 i 12

tion after aerated growth plus copper. Invariably the cytochrome c-552level was found to be raised, being generally at 10 to 30 mol per 100000 mol chlorophyll, Reconstitutions and Kinetics A remaining objection to the hypothesis of identical roles might stem from the ability of plastocyanin, but not cytochrome c-552, to restore electron transport in chloroplast fragments of ac-208(sup.), in an experiment of Gorman and Levine [18].In fact, the precise conditions of their system were that addition of 10 pM plastocyanin increased NADP' photoreduction with water as donor from 0 to 63 pmol x mg chlorophyll-' x h -',whereas addition of 2.5 pM cytochrome c-552 gave 9 pmol x mg chlorophyll-' x h-', which they attributed to a 30% level of impurity plastocyanin in their cytochrome c-552. However, this high impurity level may have been deduced from the absorption of the oxidised cytochrome at 600 nm [18,26], by being unaware that the cytochrome absorption coefficient is about 30 o/, of that of plastocyanin (see Fig. 1). Secondly, in many experiments of this type with other species it has been necessary to add 2 - 3 times more cytochrome than plastocyanin to get a comparable effect [9]. Thus Gorman and Levine's result could be interpreted as positive rather than negative. Table 4 presents data from experiments conducted to test this point, using sonicated chloroplast fragments from mutant ac-208(sup.). The first series is with electron transport through both photosystems, water being the donor and methyl viologen the acceptor (after photosystem 1). A marked stimulation was observed on addition of either protein, plastocyanin being somewhat superior. The reaction was inhibited by 3-(3,4-dichloropheny1)-1,l-dimethylurea.The second series is with only photosystem 1 operative;

P. M. Wood

the rates were much higher, but qualitatively similar. Similar experiments were conducted with wild type, but mutant ac-208(sup.)gave more dramatic stimulations [18]. This appears to be the first time that the cytochrome has been shown to stimulate a reaction involving both photosystems in a species also containing plastocyanin. Previously stimulation has only been shown with artificial photosystem 1 donors by-passing cytochromef, or by monitoring the level of photooxidation of cytochrome f in preparations with low photosystem 2 activity, for which very low rates of oxidation would suffice. Some measurements of electron transfer rates of the purified proteins were made with a dual wavelength spectrophotometer. Earlier work had shown a very fast rate of transfer from parsley cytochromef to parsley plastocyanin [23],and from Euglena cytochromef’to either Euglena cytochrome c-552 or parsley plastocyanin [ 5 ] ,but much slower rates from red-algal (Plocumium) cytochrome c-553 or Euglena cytochrome c-552 to parsley plastocyanin [5,23]. The reactivity of Chlamydomonas cytochromef with Chlamydomonas c-552 and plastocyanin was investigated for comparison, and also the reaction between c-552 and plastocyanin themselves; see Table 5. Both reactions of cytochromef‘ proved too fast to measure by this means, while the third was easily measurable and log plots yielded the rate constant shown. The conclusion is that these different cytochromesf’ from ‘green’ eucaryotes show virtually no specificity, regardless of whether the species contains plastocyanin, cytochrome c-552, or both. The reaction between the soluble cytochrome and plastocyanin is much slower in all cases, including Chlamydomonas. In rate it lies between the electron exchange reactions (which provide the one example of a reaction in which the cytochrome is far superior to plastocyanin), as expected from Marcus theory [41]. While extrapolations from such measurements to the situation in vivo require care, they suggest that cytochrome c-552 plastocyanin electron transfer is unimportant; in any case it could only be significant in the third regime studied here.

DISCUSSION The results presented in Table 1 for C. reinhardtii, wild type, and in Table 3 for mutant ac-208 and its suppressed phenotype, provide very strong evidence that plastocyanin and cytochrome c-552 constitute an interchangeable pair. This is supported by the kinetic data and reconstitutio’ns. The existence of mutant ar-208 as an acetate-requiring mutant is then seen as a consequence of the subtleties of the cytochrome c-552 versus plastocyanin regulatory system,

15 Table 4. Stimulation of electron transport in ac-208(sup.) chloroplast ,fragments by plustocyanin and cytochrome c-552 The reaction mixture contained 100 pM methyl viologen, 250 pM sodium azide (to inhibit catalase), 50 nM superoxide dismutase [38] and sonicated chloroplast fragments at 25 pg chlorophyll/ml. For (b), 10 pM 3-(3,4-dichlorophenyI)-l,l-dimethylurea and 4 mM sodium ascorbate were also added. The buffer was 20 m M potassium phosphate, pH 7, plus 20 mM KC1 and 2.5 mM MgC12. The oxygen electrode was used, at 25 “C System

Oxygen evolution pmol x mg chlorophyll-’ x h - ‘

(a) Electron transport through both photosystems ( € 1 2 0 --t methyl viologen) N o additions 0.7 pM plastocyanin 3.5 pM plastocyanin 0.7 pM cytochrome c-552 3.5 pM cytochrome c-552

+ + + +

4 19 31 11 18

(b) Photosystem 1 electron transport (ascorbate + methyl viologen) N o additions i 1.1 pM plastocyanin 7 pM plastocyanin 1.1 pM cytochrome c-552 7 pM cytochrome c-552

+ + +

108 I72 4x0 155 330

which carries on as in the wild type, although a functional plastocyanin cannot be made, and the basal rate of cytochrome c-552 synthesis (equivalent to about two molecules per thylakoid, assuming I00000 mol chlorophyll [42]) supports a minimal rate of photosynthesis. In mutant ac-208(sup.) a minor mutation (and hence a frequent one) affecting this regulatory system has led to a constitutive rate of cytochrome c-552 synthesis which is sufficient for reasonable photosynthesis. Thus the notion of equivalent roles overcomes the need to invoke a particularly unstable plastocyanin in mutant ac-208(sup.). C. mundana can be regarded as one stage further, a product of retrogressive evolution in which adaptation to anaerobic growth has resulted in a constitutive high cytochrome c-552 level and probable loss of the potential to make plastocyanin. The one point that is difficult to fit to this rationalisation is that the level of cytochrome c-552 in mutant ac-208(sup.) is quite low, about 1 mol per 5000 mol chlorophyll, which is far less than the combined levels of cytochrome e-552 and plastocyanin in wild type under any conditions. But this low level correlates well with Gorman and Levine’s observations that chloroplast fragments had to be prepared more carefully from mutant ac-208(sup.) than from wild type if activity was to be retained in reactions involving both photosystems, and that mutant ac-208(sup.) was particularly suitable for reconstitution ex-

16

Plastocyanin and Cytochrome c-552 Interrelation

Table 5. Rates of electron transfer between cytochrome f, cytochrome c-552 and plastocyanin from various organisms The rate constants in (a) were derived from log plots of pseudo first-order reactions, as in [23].The buffer was 10 mM phosphate, pH 7 , plus 80 mM NaCI, at 25 "C. The measurements were made with the dual wavelength spectrophotometer, the wavelength pairs being 409 and 422 nm, 415.5 and 422.5 nm, and 405 and 417 nm respectively Rate constant, k

Reactants reduced

Reference

oxidised

(a) Chlamydomonas reinhardtii proteins 2 1 x lo7 > 1 x 107 4 x lo5

Cytochromef Cytochromef Cytochrome c-552

Plastocyanin Cytochrome c-552 Plastocyanin

(b) Earlier work Parsley cytochrome f Euglena cytochromef Euglena cytochrome f Plocamium cytochrome c-553 Euglena cytochrome c-552

Parsley plastocyanin Euglena cytochrome c-552 Parsley plastocyanin Parsley plastocyanin Parsley plastocyanin

(c) Electron exchange reactions Bean plastocyanin Euglena cytochrome c-552

Bean plastocyanin Euglena cytochrome c-552

periments [18]. In wild type quite high activity remains after sonication [18], even though most of the soluble proteins is released, and is probably attributable to trapping of a small proportion of the plastocyanin and cytochrome c-552 in resealed vesicles. With mutant ac-208(sup.) one can imagine a similar loss reducing it to the state of true mutant ac-208. For higher plants there is often a clear lack of stoichiometry between cytochromefand P700 [43], and plastocyanin is present at several molecules per P700 [44]. These ratios are only understandable if plastocyanin is freely mobile, and there is kinetic evidence for interchain transfer at this point [45,46]. In mitochondria the analogous protein cytochrome c-550 is believed to be held on the membrane by relatively weak ionic interactions which allow two-dimensional diffusion between cytochromes c1 and aa3, while spatial considerations rule out simultaneous interactions [47]. Plastocyanin is also seemingly not involved in a ratelimiting step, which is generally reoxidation of plastoquinol [42], while the concentrations needed in reconstitutions are far less than the concentration in vivo if it is regarded as being in solution inside the thylakoids (about 600 pM if the internal volume is 10 p1 per mg chlorophyll [48]). Thus there is no obvious reason why low levels of plastocyanin and cytochrome c-552 should not confer quite high activity, and perhaps one should rather ask why chloroplasts contain so much of these proteins? They might have a function apart from electron transport, for instance in providing part of the intrathylakoid buffering at pH 4-5 [49], since there are very few other proteins known to be on this side of the membrane [50] and both have a high content of acidic residues. The answer

3.6 x 107

~ 3 1

> i x 107

PI

> 9 x lo6 5 x 105 9x104

151 ~ 3 1 151

may, however, be provided by the data in Tables 2 and 3 (and the reconstitutions in Table 4), which show a positive correlation of oxygen evolution with cytochrome c-552 content, but a marked decline in effectiveness per molecule as the level is increased. The ferredoxin-flavodoxin duality provides a useful analogy to the protein pair studied here [4,51]. Flavodoxin has been found in a number of algae and a range of bacteria. (It could not be detected in C . reinhardtii.) In many cases synthesis is stimulated by iron deficiency, with a corresponding decline in ferredoxin. Flavodoxin can substitute for ferredoxin in almost all its reactions, a remarkable fact since ferredoxin has a central role in bacterial metabolism 1511. In organisms in which as the iron level is decreased a switch occurs from ferredoxin to ffavodoxin synthesis, the change occurs before growth or synthesis of other iron proteins is markedly affected. This is exactly parallel to the response to low copper levels of plastocyanin and cytochrome aa3 in C. reinhardtii, though the usefulness of a switch from plastocyanin is more obvious since plastocyanin at 1 mol per 150 mol chlorophyll accounts for a much larger part of the copper requirement than ferredoxin does for iron. Some advantages of interchanging copper with iron have been discussed [17]. Under reducing conditions iron will be freely available as the more soluble ferrous state, but copper may be precipitated as highly insoluble sulphides. (There are very few copper proteins apart from plastocyanin that do not have oxygen as a substrate.) In a well-oxygenated environment copper will be soluble but iron is liable to be trapped as precipitated ferric hydroxide. Deficiency of dissolved iron is particularly likely in the absence of

17

P. M. Wood

organic chelating agents (oligotrophy), since although the equilibrium concentration of Fe(OH)zf, 0.1 nM at pH 7 [52], may not be insignificant, once a precipitate has formed it equilibrates only slowly with iron in solution. In the presence of organic chelators (eutrophy) a much larger pool of iron can be kept in solution, as in laboratory media, and living organisms are well equipped to extract iron from such chelates [53,54]. (Iron EDTA is used to correct iron deficiency in higher plants.) Copper is usually even more strongly chelated; for EDTA at pH 7, K’ = 3 x 1015as opposed to 1 x 1014 for ferric iron [52]. (K’ is the association constant (M - ’) for the predominant soluble species at pH 7 of metal ion (Cu”, Fe(OH)j-), EDTA (trianion), and chelate (Cu . EDTA2-, Fe . EDTA-) [52].) Although copper uptake is poorly characterised, chelated copper has been shown to be unavailable in some algal studies [55]. This could lead to iron and copper deficiencies being mutually exclusive in oxygenated media. But with C. reinhardtii plastocyanin was synthesised freely with 0.5 pM Cu2+ and 400 pM added EDTA. In this medium [H . EDTA3-] = 0.4 pM, by iterative calculation [52,56], and hence from K’ above, free CuZf is at 0.1 fM.The other anions in the medium will not greatly increase the total copper uncomplexed by EDTA [52], and even if a cell receptor can bind free Cu2’ at the diffusion controlled rate, k = 1 x 10” M-’ s-’ [57],it would only give one binding per two days per receptor. Therefore copper EDTA must be available, either directly or via an excreted chelator. The copper complex of human serum albumin, K’ = 1.5 x demonstrates that strong complexing of copper need not prevent utilization [58]. (C. reinhardtii was successfully grown with 1 mM added copper EDTA, whereas

much lower levels of CuSO4 are algicidal, but this is clearly a separate matter.) For aerated growth the results show that when copper is plentiful plastocyanin is synthesised, with only a very low basal rate for cytochrome c-552, while at lower copper levels (i.e. lower rates at which the cells can take up copper) plastocyanin synthesis slows or ceases and that of cytochrome c-552 is greatly stimulated. Neither in Boger’s study with Scenedesmus [6] nor here has it been possible to prevent plastocyanin synthesis by manipulating the iron concentration; in a full medium plastocyanin is dominant. The explanation of the basal stirring results is not so simple, since cytochrome c-552 was synthesised in the presence of abundant cupric copper in the medium; it is also quite likely that a lowish level of plastocyanin synthesis continued throughout. Perhaps an intracellular copper complex between copper EDTA and apoplastocyanin is reduced to cuprous and cannot be used, or oxygen itself may be needed, or the cells may be adapting to life with abundant ferrous iron. The regulation of cytochrome c-552 synthesis continued unchanged in mutant ac-208, which makes it unlikely that feedback from a raised level of apoplastocyanin triggers cytochrome synthesis, and the fact that apoplastocyanin could not be detected in wild type grown minus copper implies that its transcription is repressed when it is not needed. There are several other copper proteins which are potentially interchangeable with iron ones, though few studies of their interrelation. For instance, most of the bacteria in which a mitochondrial-type, 2-haem, 2-copper terminal oxidase, cytochrome 12123, is known also have alternative oxidases without copper, e.g. cytochromes al, d, o [59,60]. The copper protein

(a) ‘Green’ e u c a r y o t e s

Euglena

cytochrome c - 5 5 2

-Leu

Sambucus

plastocyanin

-?let

Rumex

plastocyanin

Phaseolus Chlorella

69

75

- S e r - Glu - As:, - Glu - I l e - V a l -

57

63

Glu

-

Asp

- Asp - Leu - Leu-

-Met

- S e r - Glu

-

Clu

- Asp - Leu - Leu -

plastocyanin

-?let

- P r o - Glu

-

Glu

- G l u - Leu - Leu -

plastocyanin

-Leu

- Ser - H i s -

Glu

- Asp - Tyr - Leu -

- Ser -

(b) The same r e s i d u e s i n b l u e - g r e e n s

Snirulina

cytochrome c - 5 5 4

-Leu

Anabaena

Flastocyanin

-Leu

Anabaena

cytochrome c - 5 5 4

-Leu

69

75

57

63

- S e r - P r o - Lys - Gln - I l e - Glu-

- S e r - H i s - Lys - Gln - Leu - Leu-

69

- Lys - P r o - Glu

-

Glu

75

- I l e - Glx-

Fig. 3. Possible sequence similarities between plastocyanin und the photosynthetic cytochrome c. The cytochrome sequences are from [63] for Euglena and Spirulina and [64] for Anabaena. The numbering of the amino acids is as in [63]. The plastocyanin sequences are from the compilation in [65]

Plastocyanin and Cytochrome c-552 Interrelation

18

azurin, in Pseudomonas and related bacteria, may well form an interchangeable pair with cytochrome c-551 [4]. Copper-zinc superoxide dismutase shares an intracellular location with iron or manganese ones in higher plant chloroplasts [61] and in its one known occurrence in a procaryote, in Photobacterium [62]. Finally, if plastocyanin and cytochrome c-552 are equivalent one might find some surface similarity by convergent evolution. The distribution of surface charges is particularly important for reactions with other proteins, and a general similarity is shown by their elution together from DEAE-cellulose. Unfortunately no green algal cytochrome has been sequenced, but Euglena cytochrome c-552 has three adjacent negatively charged amino acids with surroundings that resemble a similar grouping in eucaryotic plastocyanins (Fig. 3). The equivalent proteins in blue-greens are usually basic [4], and show a very different specificity [66,67]. Fig. 3 shows that Spirulina cytochrome c-554 and Anabaena plastocyanin resemble each other in this region, while differing markedly from the eucaryotes. On the other hand, for the only pair of proteins sequenced from a single organism, Anabaena plastocyanin and cytochrome c-554, the resemblances are less marked. So perhaps one should conclude that this approach is premature, but it will certainly be interesting to compare the tertiary structures carefully, when they are worked out. I am grateful to Professor R . P. Levine for the gift of mutant ac-208. I should like to thank Mr D. Willey for very competent technical assistance, and Dr D. S. Bendall for valuable discussions. This work was supported by a grant from the Science Research Council.

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P. M. Wood, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, Great Britain, CB2 1QW